Characterization by electrophoretic zymograms of 19 Trypanosoma cruzi clones derived from two chronic chagasic patients

1. Electrophoretic patterns of aspartate aminotransferase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, glucose-phosphate isomerase, malic enzyme and alcohol dehydrogenase have been analyzed in extracts from Trypanosoma cruzi, Tulahuén strain, 19 clones derived from isolates obtained from two chronic chagasic patients from Argentina and from Brazilian stocks Silvio X10/1 (zymodeme 1), Esmeraldo/1 (zymodeme 2), and CAN-III/1 (zymodeme 3). 2. The clones isolated from one of the patients were genetically heterogeneous. 3. Phosphoglucomutase and glucose phosphate isomerase patterns for the clones analyzed clearly differ from those of the Brazilian stocks. 4. Grouping of clones on the basis of isozyme patterns showed some correlation with that based on total DNA per organism. 5. Under the experimental conditions used, the polyacrylamide gel electrophoresis micromethod employed was advantageous over starch gel electrophoresis.     

Stability of isoenzyme and kinetoplast DNA (k-DNA) patterns in successively cloned Trypanosoma cruzi populations.

Four Trypanosoma cruzi strains from zymodemes A, B, C and D were successively cloned on BHI-LIT-agar-blood (BLAB). Twenty clones from the first generation (F1), 10 from the second (F2) and 4 from the third (F3) from the strains A138, B147 and C231 were isolated. The D150 strain provided 29 F1 and 23 F2 clones. The strains and clones had their isoenzyme and k-DNA patterns determined. The clones from A138, B147 and C231 strains presented isoenzyme and k-DNA patterns identical between themselves and their respective parental strains. Therefore showing the homogeneity and stability of isoenzyme and k-DNA patterns after successive cloning. The D150 strain from zymodeme D (ZD) showed heterogeneity. Twenty-eight out of 29 clones of the first generation were of zymodeme A and only one was of zymodeme C, confirming previous reports that ZD strains consisted of ZA and ZC parasite populations. The only D150 strain clone of zymodeme C showed a k-DNA pattern identical to its parental strain. The remaining clones although similar among themselves were different from the parental strain. Thus the T. cruzi strains had either homonogeneus or heterogeneous populations. The clones produced by successive cloning provided genetically homogeneous populations. Their experimental use will make future results more reliable and reproducible.     

Changes in Trypanosoma cruzi Kinetoplast DNA Minicircles Induced by Environmental Conditions and Subcloning

ABSTRACT. Reversible changes in kinetoplast DNA (kDNA) minicircles sequences were observed in clones of Trypanosoma cruzi strain Y, following a number of passages during exponential growth phase or after subcloning in blood-free medium. kDNA restriction patterns of clones were similar to those of the original uncloned strain, while subclones presented distinct kDNA restriction patterns. Homology experiments demonstrated strong hybridization between kDNA with the same electrophoretic mobility patterns while only weak signals were observed with kDNA of different patterns. The changes observed, which are unprecedented in T. cruzi clones, characterize transkinetoplastidy, and seem to be associated with similarly reversible changes both in zymodeme and in infectivity.     

Evaluation of a monoclonal antibody affinity purified antigen for zymodeme specific serological diagnosis of Trypanosoma cruzi infection

Theoretically, serological assays with affinity purified marker antigens can allow strain-specific diagnosis even when parasites cannot be retrieved from an infected host. A Trypanosoma cruzi antigen was purified by affinity chromatography using a zymodeme (Z) 2 specific monoclonal antibody (2E2C11). An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified antigen could discriminate between sera from rabbits immunized with T. cruzi zymodeme clones but could not discriminate between sera from mice infected with different zymodemes.     

Trypanosoma cruzi: analysis of the population dynamics of heterogeneous mixtures

Utilizing the previously reported inter-clonal differences in total DNA/organism, flow cytometry was used to analyze the population dynamics of Trypanosoma cruzi clone mixtures growing in liquid medium or vertebrate cells. The growth of clone mixtures in liquid medium can be described by unique parameters reflecting exponential growth rate (r), stationary phase population density (1/k), and the interaction between the clones (h). The relative numbers of each clone in the population change rapidly with time and the results are in quantitative agreement with mathematical models of competitive population growth. The relationship between the parameters for T. cruzi is such that, in general, there is no dynamic equilibrium with coexistence of clones with different growth rates; under all culture protocols, the faster growing clone will prevail. A computer simulation of the vertebrate cell cycle of T. cruzi suggests that clone mixtures grow relatively independently; the basic attributes of the model were substantiated experimentally. Although wide fluctuations in the proportion of each clone released occurred, the faster growing clone again predominated. Finally, these results underline the importance of working with well-defined clones in the laboratory to avoid inconsistencies and paradoxical results and stress the importance of the rapid isolation of single cell clones from clinical specimens when studying the relationship of the parasite to human disease.     

Trypanosoma cruzi: correlations of biological aspects of the life cycle in mice and triatomines.

The infection pattern in Swiss mice and Triatomine bugs (Rhodnius neglectus) of eleven clones and the original stock of a Trypanosoma cruzi isolate, derived from a naturally infected Didelphis marsupialis, were biochemically and biologically characterized. The clones and the original isolate were in the same zymodeme (Z1) except that two clones were found to be in zymodeme 2 when tested with G6PDH. Although infective, neither the original isolate nor the clones were highly virulent for the mice and lesions were only observed in mice infected with the original stock and one of the clones (F8). All clones and the original isolate infected bugs well while only the original isolate and clones E2 and F3 yielded high metacyclogenesis rates. An observed correlation between absence of lesions in the mammal host and high metacyclogenesis rates in the invertebrate host suggest a evolutionary trade off i.e. a fitness increase in one trait which is accompanied by a fitness reduction in a different one. Our results suggest that in a species as heterogeneous as T. cruzi, a cooperation effect among the subpopulations should be considered.     

Trypanosoma cruzi: biological characterization of clones derived from chronic chagasic patients. II. Quantitative analysis of the intracellular cycle

The complete intracellular cycle of five cloned stocks of Trypanosoma cruzi was quantified. Marked but stable interclonal differences were found in the length of the pre-replicative lag period (18.2-34.2 h), amastigote doubling time (8.6-21.5 h), and duration of the complete intracellular cycle (96-215 h). Strong correlations were demonstrated between these characteristics as well as to the growth rate of the epimastigote stage of the same clones grown in liquid medium. These data demonstrate that the marked heterogeneity of the natural population of T. cruzi extends to the intracellular cycle of the parasite and has important implications for our understanding of Chagas' disease.     

Trypanosoma cruzi: subtractive hybridization as a molecular strategy to generate new targets to distinguish groups and hybrids

RAPD analysis and sequences of the mini-exon and ribosomal genes show that Trypanosoma cruzi can be clustered into two phylogenetic groups-T. cruzi I and II. Herein, the Representational Difference Analysis (RDA) method was used, providing new targets specific for each group. After three rounds of RDA hybridizing F strain (tester) with Y strain (driver) and vice-versa, an F-specific (F#30) and Y-specific (Y#22) clone were obtained specifically recognizing isolates from Amazonas (T. cruzi I) and Piauí (T. cruzi II). These segments corresponded to an unspecified protein (F#30) and a trans-sialidase (Y#22). Analysis of the F#30 sequence in T. cruzi I, T. cruzi II and zymodeme 3 samples displayed negligible specific differences that distinguished each group. In addition this F#30 gene has great potential as a hybrid marker.     

Comparative analysis by polymerase chain reaction amplified minicircles of kinetoplast DNA of a stable strain of Trypanosoma cruzi from São Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of S?o Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicircles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment length polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.     

Trypanosoma cruzi I and Trypanosoma cruzi II: recognition of sugar structures by Arachis hypogaea (peanut agglutinin) lectin

Epimastigote culture forms of different isolates of Trypanosoma cruzi from different mammal hosts, humans, and vectors were tested with FITC-conjugated peanut agglutinin lectin (PNA-FITC). The parasites maintained in axenic medium, liver infusion tryptose. were evaluated by flow cytometric analyses; whereas T. cruzi I (Tcl), which is associated with the sylvatic transmission cycle, was labeled in high percentages with PNA (88-99.2%), T. cruzi II (TcII) (parasites associated with domiciliar cycle) and T. cruzi, zymodeme 3 (Tc/Z3) (also associated with the sylvatic cycle) were labeled in low percentages (TcII, 0-26% and Tc/Z3, 0-12.6%). It was demonstrated that it is possible to differentiate the 2 main T. cruzi subpopulations, TcI and TcII, using Arachis hypogaea. These results also showed a higher variability in TcII in terms of PNA binding.     

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.     

Biological characterization of Trypanosoma cruzi strains from different zymodemes and schizodemes.

The development in C3H mice of thirteen strains of Trypanosoma cruzi belonging to different zymodemes and schizodemes was studied. Host mortality, virulence, histiotropism, parasitemia and polymorphism of the parasites were recorded. The strains were grouped into: a) high virulence--causing 100% mortality and characterized by predominance of very broad trypomastigotes in the bloodstream at the end of infection; b) medium virulence--causing no mortality and with a predominance of broad trypomastigotes; c) low virulence--causing no mortality with blood forms not described due to the very low parasitemia. During 18 months maintenance the parasitemia curves were kept constant for all strains except one. A direct correlation between either zymodeme or schizodeme and experimental biological properties of T. cruzi strains was not found. However, the parasitemia was subpatent and patent for strains from zymodeme C and the others respectively. Furthermore the high virulence seems to be related to one of two schizodemes found within zymodeme B strains. All strains presenting patent parasitemia independent of shizodeme and zymodeme showed a myotropism towards heart and skeletal muscle with variable inflammatory intensity. The present study confirmed the heterogeneity found by isoenzyme and k-DNA patterns among the strains of T. cruzi isolated from chagasic patients in Bambuí, Minas Gerais State, Brasil.     

Quantitative differences in the expression of a 72,000 molecular weight cell surface glycoprotein (GP72) in Trypanosoma cruzi zymodemes

A high affinity monoclonal antibody, 8G2 B9, was used to assess the expression of a 72,000 m.w. glycoprotein ( GP72 ) in isoenzyme-typed T. cruzi strains ( zymodemes ). Western blotting analysis of T. cruzi clones showed that 8G2 B9 bound strongly to GP72 and also suggested that this antigen was absent or weakly detectable in T. cruzi zymodeme 1 (Z1) strains. Purified 8G2 B9 was radiolabeled with 125I and used in an inhibition radioimmune binding assay to compare the quantities of GP72 in different zymodemes . Ninety-six T. cruzi strains were assayed, of which 36 were Z1, 36 were Z2, five were Z3 , and 19 were Z2 (heterozygous). Most (64%) Z1 strains lacked detectable GP72 , whereas this antigen was always detected Z2 and Z2 (heterozygous) strains. There was an 18-fold difference between geometric mean values for the quantities of GP72 (expressed as nanograms per milligram total cell protein) in Z1 and Z2 strains (Z1, 36 ng/mg; Z2, 639 ng/mg; p less than 0.001). There were also significant differences between the geometric mean values for Z2 and Z2 (heterozygous) strains, i.e., 639 ng/mg and 1648 ng/mg, respectively (p less than 0.001). GP72 was detected in four of the Z3 strains in quantities ranging from 740 to 3640 ng/mg. The absolute amounts of antigen in GP72 -positive strains were low, comprising less than 1% of the total cell protein. The specificities of two other anti- GP72 monoclonal antibodies, 7C6 D7 and WIC 29.26, were compared with 8G2 B9. Both antibodies completely inhibited the binding of 8G2 B9 to GP72 in solid phase immunoassays, suggesting that they reacted with the same antigenic determinants. The results show that monoclonal antibody-based assessments of the expression of GP72 correlate with zymodeme classification, and they also suggest that the monoclonal antibodies recognize major antigenic determinants on GP72 . It should be possible to use 8G2 B9 as an immunologic marker to additionally investigate the clinical significance of T. cruzi zymodemes and the biologic significance of GP72 .     

Trypanosoma cruzi: quantitative studies of development of two strains in small intestine and rectum of the vector Triatoma infestans

This paper describes the development stages and numbers of flagellates of two strains of Trypanosoma cruzi living in the small intestine and rectum of the insect, Triatoma infestans, during the first 12 weeks postinfection (pi). Mainly epimastigotes and occasionally amastigotes and final trypomastigotes developed in the small intestine but after starvation periods of 3 or 4 weeks higher percentages of spheromastigotes including their transitional forms to/from epimastigotes were found. In the rectum, the percentage of final trypomastigotes increased in two steps; the second, but not the first, correlated with the development of intermediates originating from epimastigotes. For both strains the total number in the small intestine increased during the first 8 or 9 weeks, although there were reduced numbers when the bugs had starved for 3 or 4 weeks. In the rectum the numbers increased up to 10 weeks pi; only about 25% of these lived in the lumen, the others were located at the rectal wall. In small intestine and rectum the "Chile 5" strain of T. cruzi (zymodeme 1) nearly always reached higher population densities than the "Chile 7" strain (zymodeme 2).     

Genetic Heterogeneity in Trypanosoma cruzi Strains From Naturally Infected Triatomine Vectors in Northeastern Brazil: Epidemiological Implications

Eighteen Trypanosoma cruzi strains isolated from naturally infected triatomines were studied genetically. The majority of the strains were from Triatoma brasiliensis, the principal vector of Chagas disease in the northeast of Brazil. Multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) analyses were used to investigate the genotypic diversity and the spread of the T. cruzi genotypes in different environments. MLEE clearly distinguished two distinct isoenzyme profiles, and RAPD analysis revealed 10 different genotypes circulating in rural areas. The strains could be typed as isoenzyme variants of the T. cruzi principal zymodeme Z1 (T. cruzi I). An effective program of epidemiological vigilance is required to prevent the spread of T. cruzi I strains into human dwellings.     

Trypanosoma cruzi: exploring the nuclear genome of zymodeme 3 stocks by chromosome size polymorphism

Chagas disease is emerging in the Brazilian Amazon. We evaluated the position of eight zymodeme 3 isolates from Amazonian sylvatic vectors and one human case in relation to Trypanosoma cruzi I and II major groups and hybrid strains by chromosome size polymorphism. Nineteen isolates were analyzed by mapping nine coding sequences on chromosomal bands (0.6-3.3Mbp). Numerical analysis was based on the absolute chromosomal size difference index (aCSDI). A dendrogram was obtained applying the minimum evolution criterion and considering the aCSDI values to estimate the branch lengths. The isolates were distributed in four groups. Group A clustered hybrid isolates; Groups B and C, T. cruzi II and T. cruzi I isolates, respectively. Seven Z3 stocks were clustered in Group D, which showed low intra-group diversity and was the most divergent. The proportion of two different-sized homologous chromosomes was determined. Wild vectors harboring Z3 stocks constitute a potential reservoir of human infection in the Amazon.     

Identification of Trypanosoma cruzi Zymodemes by Kinetoplast DNA Probes

Analysis of zymograms of extracts of Trypanosoma cruzi isolated from different hosts in Argentina allowed characterization of 12 zymodemes or "isozymic strains," only six of which were found in human patients. Two of these six zymodemes (Z1 and Z12) were widely distributed and found in more than 80% of human patients. These two "major natural clones" differed significantly in pathogenic activity. Because the groupings obtained by studying enzymes and kinetoplast DNA (kDNA) were similar, it is possible to identify the zymodeme by analyzing kDNA. A 290-bp fragment was amplified by PCR using primers for the sequences flanking the hypervariable regions of kDNA minicircles. Labeled probes for this fragment, prepared from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified kDNA from parasite stocks, allowing identification of zymodemes.     

Trypanosoma cruzi: isolation and characterization of two clones derived from neonatal mice

The Tulahuen strain of Trypanosoma cruzi was cloned in 15 C3H/Anf neonatal mice. Ten of these 15 neonates became parasitemic before the 12th day and died before the 19th day after the inoculation of a single bloodstream trypomastigote. Two clones were selected and maintained, while the other isolates which did not grow in a liquid metacyclic stage culture (LMC) medium were eventually discarded. The kinetics of in vitro growth and transformation from epimastigote to metacyclic trypomastigote of these two clones were characterized in LMC medium at 27 degrees C. Infectivities for vertebrate cells in vitro were retained by these two clones during the period of cultivation. The tropism for brain, heart, lungs, esophagus, stomach, large intestine, liver, pancreas, spleen, lymph nodes, kidneys, bladder, and skeletal muscles was also examined in the mice. The communication describes the establishment and characterization of T. cruzi clones. The utilization of these cloned parasites should produce some advantages in generating reproducible data in investigations.     

Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice

Thirty-one Trypanosoma cruzi isolates from Chile, Peru, and Bolivia were studied in their capacity to differentiate in vitro from epimastigotes to metacyclic trypomastigotes on TAU-3AAG medium. Zymodeme 1 parasites displayed the best level of differentiation, which ranges from 60 to 90% depending on the isolate. Zymodeme 2 parasites exhibited highly heterogenous differentiation rates. This differentiation method permits the obtention of large amounts of metacyclic trypomastigotes from zymodeme 1 parasites. Metacyclic trypomastigotes obtained in vitro were infective to nude Balb/c hybrid mice. Zymodeme 1 parasites produced high parasitemias in this murine model; in contrast, zymodeme 2 parasites displayed lower parasitemias. Of a total of 27 T. cruzi isolates, 20 proved to be infective to mice, 12 gave enough parasites for further studies, and 8 of these were used for biological characterization. Results are compared with the infective clone Dm28 and Tulahuén strains maintained since 1954 in mice.     

Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium

The isolation of biological clones of Trypanosoma cruzi by microscopically dispensing individual organisms or by serial dilution is laborious and time consuming. The inability to resolve mixed T. cruzi infections, from vectors and hosts, and to isolate clones of slow growing genotypes by efficient plating on solid media, has hindered characterisation studies and downstream applications. We have devised and validated a sensitive, solid medium plating technique for rapid in vitro isolation of clones representative of all the recognised T. cruzi lineages (TCI, TCIIa-e), including the slow growing strain CANIII (TC IIa) and Trypanosoma rangeli, with high plating efficiencies. Furthermore, the method is effective for the isolation of clones directly from silvatic triatomine bugs and from experimentally infected mice harbouring mixed infections, allowing resolution of multiclonal infections from varied sources.